I. Field of the Invention
The invention relates generally to protein purification; more specifically to enzyme purification. In particular, the invention provides a preparation and method for purification of the enzyme, flavin adenine dinucleotide (FAD) synthetase, preferably from microbial origin. FAD synthetase is formally designated ATP: FMN adenylyl transferase (EC 2.7.7.2) by the International Congress of Enzyme Nomenclature.
II. Description of the Prior Art
In general protein purification methods are applicable to enzyme purification. Such generally applicable methods are available in many literature references, see for example Biochemistry 2nd Ed., Lehninger, A., Worth Publishers, Inc. (1975). Affinity chromatography is one generally applicable method of protein purification. One variant of affinity chromatography is dye-ligand chromatography in which synthetic textile dyes are used as immobilized ligands. Use of these dyes in enzyme purification has gained wide acceptance since the late 60's. Many applications of dye-ligand chromatography have been made to the purification of adenosine triphosphate (ATP) binding enzymes. For example, Thompson et al, 72, Proc. Nat. Acad. Sci., USA, 669-672 (1975) described the purification of adenylate kinase, an ATP-binding enzyme, on Cibacron.RTM.-Blue Sepharose with subsequent elution with ATP or adenosine diphosphate (ADP). However, few applications have been made to the purification of flavin-binding enzymes, such as FAD synthetase. In one case, Pompon et al., 110, Eur. J. Biochem., 565-570 (1980), purified the apoenzyme, apo(cytochrome b.sub.5 reductase) on immobilized Cibacron.RTM.-Blue F3GA with subsequent elution with FAD or flavin mononucleotide (FMN). The apoenzyme is the active enzyme without its activating cofactor, FAD. Purification of the apoenzyme was used since most enzymes bind flavin too tightly to allow successful purification using dye-ligand chromatography with subsequent competitive elution. Moreover, the binding mechanism between the dyes and the enzymes is not fully understood and varies significantly from enzyme to enzyme and from dye to dye.
One measure of enzyme purity is its specific activity. As used herein the specific activity calculated for FAD synthetase is based on the ability of the enzyme to catalyze the reaction ##STR1## The units of specific activity used herein are nanomoles per minute per milligram, i.e., the enzyme preparation can produce the stated number of nanomoles of product, FAD, per minute per milligram of protein in the enzyme preparation used, at 25.degree. C.
Since ATP and FMN are coenzymes in many biological reactions, their presence is an indication of the presence of biosystems in a sample. Further, the generation or consumption of these coenzymes in reactions can be used to diagnose particular clinical states. However, the concentration of ATP in biological fluids is often only 10.sup.-9 moles/liter (nanomolar) to 10.sup.-12 moles/liter (picomolar) so that highly sensitive assays are required. Enzymatic assays provide an advantage over stoichiometric assays since one molecule of enzyme can catalyze the formation of thousands of molecules of product. In order to take full advantage of this phenomenon, the enzyme preparation used should have as high a specific activity as possible.
Spencer, et al., Biochemistry, 15:5, 1043-1053 (1976) isolated FAD synthetase from a culture of Brevibacterium ammoniagenes (ATCC 6872). Their procedure involved (a) centrifuging broken bacterial cells; (b) separating the liquid fraction; (c) precipitating the protein in the liquid fraction with ammonium sulfate; (d) applying the precipitated protein in solution to a Sephadex.RTM.-G-100 column and eluting. The FAD synthetase preparation obtained had a specific activity of 4.9 nanomoles per minute per milligram at 37.degree. C. with radioactive flavin mononucleotide as the substrate.